Cartridge for containing a specimen sample for optical analysis

ABSTRACT

An improved cartridge for holding a fluid sample with a small volume is disclosed herein. The cartridge has a test chamber and a vestibule through which the test fluids are inserted into the test chamber. Improved grips are flared-out to aid manipulation. The handle portion is reinforced to prevent flexing, and a prefabricated trough along the edge of the land surface prevents introduction of the adhesive into the region for analysis. The cartridge has a stopper having a dual sealing mechanism, which seals the test chamber inlet between the vestibule and the test chamber, and the mouth of the vestibule so that when the stopper is in place, the test chamber is closed to the admission of air or other contaminants. The vestibule is similarly closed against escape of the overflow from the test chamber. The stopper is composed of a single elastomer. An improved locking mechanism has two flexible walls on either side of the handhold that locks into their respective keepers on the cartridge to provide a secure lock. Additionally, an improved method for loading the cartridge allows for an even distribution of magnetically responsive particles contained within a fluid sample to be viewed in an array through the optically clear land surface portion of the cartridge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 10/303,309, filed Nov. 25,2002 now U.S. Pat. No. 7,011,794, now allowed.

FIELD OF THE INVENTION

The present invention is an improvement upon an earlier filedapplication relating to a cartridge having a chamber for containing aspecimen sample used in optical analysis (PCT/US02/04124; U.S. Pat. No.6,861,259; U.S. Ser. No. 10/988,057). One improvement enables the use ofthe cartridge in a manner that is more practical and less expensive foranalysis of small biological samples, particularly blood. Anotherimprovement enables a more efficient and uniform distribution ofmagnetically labeled target components within a sample prior toanalysis.

BACKGROUND OF THE INVENTION

When performing optical analysis on specimens in liquid suspension, itis customary to discard the specimen after analysis. For rare cells,however, it is often desirable to preserve the liquid specimen forfurther testing or for use in further procedures. In certain procedures,it is desirable to select a specimen of a relatively small sample (lessthan 400 ul) from larger volumes (up to 100 ml), and it is likely thatthe volume of the specimen, which may be extracted from such a sample,is limited, making it especially important to avoid destruction of thespecimen or any substantial part thereof. It is likewise important toavoid contamination of the specimen and to avoid conditions leading todeterioration of the specimen or impairment of analysis.

One cause of inconsistent optical analysis from sample to sample is alack of uniform distribution of the magnetic particles and themagnetically labeled target components within the fluid sample due togravitational forces. This pooling affect occurs when the sample isfirst placed into the cartridge chamber. Unless the chamber is perfectlyhorizontal, the magnetic particles, together with the magneticallylabeled target components tend to settle toward the lower portions ofthe chamber. When the chamber is loaded in the vertical position, apreferred loading position, the magnetic particles and the magneticallylabeled target components settle toward the distal portion of thechamber. Consequently, the magnetic particles and the magneticallylabeled target components do not distribute uniformly across the opticalwindow, causing the inability to perform optical analysis.

Another issue in the development of the cartridge and stopper is toprovide a structure that is relatively inexpensive to manufacture(PCT/US02/04124), yet functions satisfactorily in preserving the sample.Construction, from two different plastic materials such as thatdescribed in the stopper component of the parent application, areexpensive to manufacture and complicated in their construction. Sincespecimen samples are stored in a manner to prevent deterioration, therealso is a need to maintain the locking feature so as to provide a properseal against contamination and air. Thus, a stopper that wouldincorporate the benefits of a single elastomer composition together witha locking mechanism to minimize deterioration of the sample is needed.

In conjunction with a need to improve sample storage for opticalanalysis, there is a need to increase the ease and speed ofmanipulations of the cartridge. A better grasp on the cartridge wouldprevent unnecessary cartridge flexing that could allow air to enter thetest chamber, and would provide more secure transfer of the cartridgefrom the loading position to the platform for analysis.

SUMMARY OF THE INVENTION

As can be seen from the discussion, above, there is a need to furtherimprove upon select aspects of US Patent Application PCT/US02/04124. Thepresent application describes a collection chamber designed to isolate asmall sample for analysis and to preserve the sample for additionaltesting. Specifically, this collection device enables the exclusion ofair as bubbles or other forms from the sample chamber, but does notprovide a low cost chamber with uniform analyte distribution foranalysis.

U.S. Pat. No. 5,246,669 discloses a sampling device for collecting asmall sample and mixing it with a test liquid. In this patent, thedevice provides a pickup device, which extracts a small sample from alarger quantity of solid or semi-solid material to be tested. The deviceseparates the small sample from the residue and isolates the residue soas to avoid contamination of the small sample or the surroundingatmosphere. The device does not provide for salvaging either of the testsample or the residue and does not have any provision for excluding airin the form of bubbles from the test liquid.

In addition to the need to redesign the cartridge and stopper, improvedoptical analysis is also obtained in the manufacturing process of thecartridge of the invention. There is a need to manufacture a cartridgethat allows for a consistently clear and uniform area for viewing. Thesample would circumvent problems in the analysis of magnetically labeledtarget components, particularly with respect to those target componentslocated close to the contact area between the cartridge dome and theoptically clear land surface interface. More specifically, interferencefrom adhesive used to join the optically clear land surface with therest of the cartridge results when the adhesive spreads onto the opticalpath during the manufacture of the chamber. Thus, there is a need todevelop a process for affixing the optically clear land surface in a waythat prevents the introduction of adhesive onto the optical analysisregion.

Improving the ability to manipulate a chamber containing magneticallylabeled target components within a sample coupled with the ability todisplay them in a uniform manner, all in a low cost manner, enables thepresent invention to provide a consistent platform for optical analysis.

Accordingly, the present invention provides an improvement to a novelcartridge for use in optical analysis of specimens. As with the originalcartridge, the improved version has a test chamber that contains thespecimen for subsequent procedures, provides a platform for opticalanalysis of the specimen without loss of any substantial part thereof,and enables the specimen to be retained in the cartridge test chamber inthe absence of air bubbles or other contaminants. The cartridge is alsoused in a method for handling specimens enabling the specimen to bepresented in a test chamber for optical analysis without the risk forloss of any substantial part of the specimen and without the risk ofinclusion of air bubbles or exposure to other deteriorating conditionsin the test chamber with the target components evenly distributed foroptical analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the cartridge and receptaclehaving a pair of opposed magnetic poles made in accordance with thepresent invention.

FIG. 2 is a front view of a cartridge embodying the present inventionand the associated improvements with its stopper in place to illustratethe engagement of the stopper with the body of the cartridge.

FIG. 3 is a view similar to FIG. 2 showing the stopper removed from thebody of the cartridge.

FIG. 4 is a cross-sectional (A), magnified cross-sectional (B), andlongitudinal-sectional (C) view. FIGS. 4A and 4B are cross-sectionalviews taken on the line 4A-4A of FIG. 3. FIG. 4B is an enlargedcross-sectional view of the adhesive trough on the edge of the shelfportion of the body juxtaposed to the optically clear edge of the landsurface. FIG. 4C is a longitudinal-section taken on the line of 4B-4B ofFIG. 3.

FIGS. 5A, 5B, AND 5C are sectional views of the cartridge in loadingposition illustrating the cooperation between the cartridge body and thestopper to entrap the specimen in the cartridge in the absence of air.FIG. 5A shows the stopper removed with the specimen in place within thechamber and the vestibule. FIG. 5B is a view similar to FIG. 5A showingthe penetration of the probe of the stopper into the buffer within thevestibule. FIG. 5C shows the stopper in place closing both the portbetween the chamber and the vestibule and the upper end of thevestibule.

FIG. 6 is a perspective view of the stopper shown in FIGS. 2-5.

FIG. 7 is an enlarged fragmentary sectional view of the cartridge withthe stopper operatively engaged with the body of the cartridge.

DETAILED DESCRIPTION OF THE INVENTION

The device of the present invention has been improved to limitsignificant settling of target components prior to loading, thusenabling a uniform distribution of the array of magnetically responsiveparticles and magnetically labeled target components along the viewingsurface. Because the biological sample is loaded into the cartridge inthe presence of a magnetic field, an equal distribution of magneticallylabeled target components is ensured. Loading is accomplished by placingthe cartridge vertically within a magnetic field prior to sampleloading. The sample is then introduced into the cartridge chamber,closed with a stopper, and maintained until analyzed. Optical analysisis completed without settling and with a uniform distribution of targetcomponents that remains consistent from sample to sample.

As with the cartridge from the parental application, the specimen samplecan be introduced into the test chamber of the cartridge along with abuffer solution in which the respective properties of the specimen andthe buffer solution are such as to provide an interface separating thebuffer solution from the sample. The sample chamber is elongated with aport at one end. Preferably, the buffer solution is of a density lessthan or equal to the density of the sample so that when the chamber isdisposed with the ported end upright, the buffer solution is positionedabove the interface and the sample is positioned below the interface.During the filling of the chamber, any air or other contaminants, whichare less dense than the buffer solution, are allowed to gravitateupwardly through the buffer solution towards the port in the upper endof the chamber. The chamber is designed with a vestibule containing anoverflow reservoir capable of being sealed both from the test chamberand the exterior environment. A stopper is provided for sealing bothends of the vestibule through its attached probe, which extends into thebuffer solution above the interface. As the stopper, through the probe,is engaged with the port of the test chamber, the probe displaces thebuffer into the vestibule. The stopper now forms a primary seal, whichcloses the test sample chamber from the vestibule. Subsequently, thestopper forms a second seal to close the outside entrance of thevestibule to prevent escape of the buffer from the vestibule. Thevestibule thus serves as an overflow receptacle. The first seal closesthe test chamber after any deleterious air bubbles in the chamber havemigrated into the vestibule. Thereafter, the second seal closes theoverflow chamber to retain the buffer solution against loss with thesimultaneous engagement of the stopper through an improved clipmechanism.

Improvements on the stopper results in reduced expense and increasedease of cartridge handling. The improved stopper is composed of a singleelastomer with a preferred durometer value of about 60 to 90. Thestopper is locked into position in the cartridge by flexible walls sothat when the second seal closes the overflow chamber the walls arecompressed until they snap outwardly to position against theirrespective keeper elements. This interaction allows the stopper to lockinto place, making stopper alignment with the chamber easier and makingthe stopper much more difficult to remove after filling.

When in place, the stopper and handle permit the cartridge to be gripedand manipulated into position with the optical detecting device in anorientation which positions the test chamber so that the target cellsare in a suitable position within the field of observation for thedetection equipment.

Grips on the cartridge used for manipulating the cartridge into positionhave been improved by flaring-out the inward taper to allow a moresecure grasp and better control. Accordingly, the cartridge may beeasily mounted in a microscope or other detection equipment to allowpositioning of the target components in the biological sample within theequipment's sample observation field. Within the handle portion andbetween the grip areas, the cartridge has improved support to preventunintentional flexing thereby affecting the established seals betweenthe stopper and cartridge.

Finally, a novel type of manufacture for the attachment of the opticalland surface to the plastic portion of the cartridge is described. Morespecifically, a plastic edge is in contact with an optically clear landsurface, which is usually glass. The edge is shaped as a small shelf toseat the land surface. The shelf spans the entire area to be in contactwith the land surface. A small trough is positioned along the shelf, butwithin the area to be in contact. A binding adhesive is introduced intothe trough and along the entire contact region. The binding adhesive isan optically clear adhesive that is non-fluorescent and unable to reactwith the sample. When the optically clear land surface is placed incontact with the shelf, the adhesive is channeled away from the opticalpath. This allows for a more reproducible and for a more consistentobservation during optical analysis. While any adhesive with thecharacteristics described above will work, adhesives can be of apressure-sensitive type, heat cured type, or activated by ultravioletlight.

Optical analysis of biological samples, especially rare cells, requiresa method and means to maintain the specimen in a condition that wouldallow inexpensive and reproducible. The improved sample cartridge andstopper, when used together, are designed to avoid contamination of thebiological sample, but with an improved design and manufacture whichallows for better control during any manipulation prior to opticalanalysis, uniform distribution of magnetically labeled target componentsfor optical analysis, and at a lower cost to produce.

One improvement in the preparation of the sample is to load and storethe cartridge assembly, either the parental or improved version, withinan applied magnetic field prior to sample filling and optical analysis.The cartridge is positioned vertically before loading within an appliedmagnetic field in preparation for sample loading. The sample isintroduced into the cartridge chamber within the magnetic field,resulting in the prevention of any settling of magnetically responsiveparticles and any magnetically labeled target components. Accordingly,the magnetically labeled target components are arranged in an evenlydistributed, which is consistently displayed from sample to sample. Themagnetic field prevents these same magnetically labeled targetcomponents from clustering within one region of the cartridge prior tooptical analysis. Thus after insertion of the stopper into thecartridge, the magnetically labeled target components of the biologicalsample will remain stably positioned along the flat land surface of thecartridge until removed from the magnetic field after analysis.

The cartridge and stopper of the improved invention are particularlyadapted for use in a detecting apparatus such as shown in U.S. Pat. No.6,013,532, which issued to Liberti et al. on Jan. 11, 2000, anddescribed in a paper entitled “Optical Tracking and Detection ofImmunomagnetically Selected and Aligned Cells” by Arjan G. J. Tibbe etal., Published in Nature Biotechnology, Vol. 17, December 1999, pp1210-1213, both of which are incorporated by reference herein. Thecartridge and receptacle, shown schematically in FIG. 1 of the patent,is effective to immobilize magnetically labeled target entities, such ascells, within a fluid medium for observation, analysis or manipulation.The magnetically labeled target entities are deposited in a test chamberwhere they are manipulated by a magnetic field to dispose themagnetically labeled target entities in a monolayer along a wall of thetest chamber. A discussion of automated magnetic separation techniquesis included in U.S. Pat. No. 5,985,153 which issued to Gerald J. Dolanet al. on Nov. 16, 1999, and in a paper entitled “Cell Analysis SystemBased on Immunomagnetic Cell Selection and Alignment Followed ByImmunofluorescent Analysis Using Compact Disk Technologies” by Arjan G.J. Tibbe et al., published in Cytometry, 43:31-37 (2001), both of whichare also incorporated herein by reference.

Referring to FIG. 1, an improved cartridge embodying the invention isshown at 21 mounted in a receptacle 20 having a pair of opposed magneticpoles, 22 and 23 which have a gap formed therebetween. In theillustrated detecting apparatus, the receptacle 20 is positionedhorizontally in the path of the optical system of the apparatus with thegap upwardly, but for other applications the receptacle may bepositioned vertically. In FIG. 1, the lower surfaces of the poles 22 and23 are tapered toward the gap so that magnetic field applied to thechamber is non-uniform and has a substantially vertical gradient effectdirected toward the gap transverse to the longitudinal axis of thecartridge 21 to urge magnetically-responsive particles and magneticallylabeled targets within the chamber towards the wall of the cartridgewhich is substantially co-planar with the gap. The magnetically labeledtarget entities are displayed as an orderly monolayer on the interiorsurface of the test chamber, and an automated observation system can beconfigured to provide relative motion between the cartridge and thelight-gathering elements of the observation system in order to tract thecollected magnetically labeled target entities for automatedenumeration, which can include spectral analysis of light emitted,absorbed or scattered by the collected magnetically labeled targets.

The complete system comprises optical tracking beam analysis componentssimilar to those employed for reading compact discs known in the audioand data storage arts (see PCT/US02/04124). Briefly, a pair of laserdiodes generates parallel beams of light. One beam is employed by theanalysis system (not shown) for locating and tracking lines of thetarget entities. The other beam is used for detecting the presence ofcollected target entities adjacent to a located line. Relative motionbetween the cartridge and the optical elements of the analysis system isprovided by a mechanical translation unit (not shown). Coordination ofthe functions of the analysis system is provided by a microprocessor(not shown). The tracking beam, which is reflected by dichroic mirrorthrough the aperture is focused upon the upper surface of the cartridgeby an objective lens. The dichroic mirror 7 through the dichroic lensand the objective lens reflects the detecting beam.

Light reflected by the tracking lines and the target entities will betransmitted through dichroic mirrors and toward a photo detector (notshown). The detector generates a data signal, which is fed to themicroprocessor for the unit, as described more fully in theabove-mentioned U.S. Pat. No. 5,985,153, to control the translation ofthe unit and process the data provided by the detector.

The improved cartridge 21 may also be used in other detecting apparatussuch as a microscope, as described in the above-mentioned U.S. Pat. No.6,013,532, in which the stage is designed to receive the receptacles 20so as to position the surface of the cartridge in the light path of themicroscope. As noted above, the orientation of the test chamber may behorizontal, vertical or at any angle determined by the instrumentationof the detecting apparatus.

As shown in the orientation of FIG. 1, the improved cartridge 21 has adomed body portion 51 having outwardly projecting glides 52 and 53 onopposite sides thereof. While holding the cartridge by the grips 37 38,the glides 52 and 53 are designed to slide into guide ways 54 and 55 inthe receptacle so that the domed body portion of the cartridge underliesthe lower surfaces of the poles 22 and 23. Intermediate between theguide ways 54 and 55, the receptacle has a slot 56 providing an opticalpath through the bottom of the receptacle. The optical path registerswith the longitudinal centerline of the cartridge when the cartridge isinserted into position within receptacle 20.

FIG. 2 shows the cartridge 21 in front view embodying the presentinvention and the associated improvements with its stopper in place toillustrate the engagement of the stopper with the body of the cartridge.The stopper is shown with plug 74 sealingly engage. With the stopperfully inserted, the plug 74 closes the proximal end of the vestibule.The stopper has an improved handhold 75 and an improved lockingmechanism composed of two flexible walls 76, 76. The flexible walls 76,76 flank the plug 74 and engage their respective keeper elements 77, 77also improved and located in the handle portion 61 of the cartridge. Theimproved keeper elements are supported at the top of the handle portionand laterally along the sides of the keeper elements. This providesextra rigid support when engaged as shown.

FIG. 3 shows the cartridge 21 in front view embodying the presentinvention and the associated improvements with its stopper removed toillustrate the difference when not engaged with the body of thecartridge

The improved cartridge is formed from a non-magnetic inert material,such as polycarbonate, polystyrene or acrylic with no fluorescentadditives and is formed to provide a rigid chamber, which may bemanipulated into and out of the optical path of the optical analysissystem. A sectional view with the stopper removed shows the cartridgewith flanking grips 37, 38 and a centrally located flat land surface 62formed from optically clear material such as glass and affixed at thetop of the dome 51 (FIG. 4A).

A procedure for affixing the optically clear land surface 62 to thecartridge is incorporated into the embodiment to provide better adhesion(see FIG. 4), a more consistent orientation of the land surface 62 withrespect to the viewing aperture, but most importantly less interferencewith optical analysis due to excess adhesive in the chamber area. Theprocedure allows for a more even and controlled application of theadhesive along the interface between the land surface 62 and the top ofthe non-magnetically inert dome 51. More specifically, the improvedaffixation prevents the adhesive from spreading in both lateraldirections by forcing adhesive away from the chamber area.

Basically, the improvement is in the geometry of an adhesive trough 12,which is designed to channel the adhesive away from the chamber areawhen the flat land surface is attached (FIG. 4B). The molded inertmaterial of the cartridge dome is shaped as a small shelf 15 at the edgeto form the contact area with the flat land surface 62. A small trough,filled with adhesive, runs along the edge. The trough spans the entirecontact area so that when the flat land surface is placed on the edge,contact is made with the adhesive. The adhesive is allowed to wick downthe trough and across the contact area. This area is then exposed to UVlight, heat cured, or treated by any means to securely bond the edge tothe land surface. The preferred embodiment is to use a UV light adhesiveto bond 0.5 mm thick glass cut to tight tolerances with molded plastic.The molded plastic must be non-fluorescent, rigid, optically clear, andnon-reactive with the sample. Typical examples are polycarbonate orpolystyrene.

The land surface 62 is optically clear and permanently affixed to thecartridge. Thus when positioned in the receptacle 20, the test chamber63 is aligned with the aperture 56 of the receptacle along the lightpath of the detecting apparatus in which receptacle 20 is mounted. Thisprovides for an optimum analytical viewing surface.

The test chamber 63 is closed at the distal end remote from the handleportion 61 and has an inlet opening 65 at the proximal end adjacent thegrips 37, 38 (FIG. 5A). The inlet 65 is positioned in the center of theend wall of the test chamber 63 at the proximal end so that when thecartridge is disposed vertically for filling the inlet opening 65 is atthe uppermost part of the chamber 63. The body of the cartridge providesa vestibule chamber 66 having an enlarged mouth 67 at its entrance end.The vestibule chamber 66 communicates with the test chamber 63 throughthe inlet opening 65. Between the mouth 67 and the inlet 65, thevestibule 66 provides an overflow reservoir, as described more fullyhereinafter.

The test chamber 63 is adapted to be closed by the plunger 71 of animproved stopper having a probe 72 adapted for sealing through anengagement with the inlet 65 of the test chamber 63. Rearwardly of theprobe 72, the stopper has a ribbed stem 73. The ribbed stem terminatesinto a plug 74, which is adapted to sealingly engage in the mouth 67 asthe stopper is fully inserted through the vestibule 66. When fullyinserted, the plug 74 closes the proximal end of the vestibule 66.Beyond the plug 74, the stopper has an improved handhold 75 and animproved locking mechanism composed of two flexible walls 76, 76. Theflexible walls 76, 76 flank the plug 74 and engage their respectivekeeper elements 77, 77 also improved and located in the handle portion61 of the cartridge. The improved keeper elements are supported at thetop of the handle portion and laterally along the sides of the keeperelements. This provides extra rigid support when engaged. When theflexible walls of the stopper 76, 76 are engaged, they are compressed asthey are pushed by the improved keeper elements 77, 77, but then snapoutwardly when finally in place.

The improved cartridge has a handle portion 61 with associated gripsenabling the insertion and removal of the cartridge into and out fromthe receptacle. The handle portion 61 has also been improved by flaringthe side grips 37, 38 located orthogonal to the first face. The griparea is made to flare away from the radial axis of the cartridge. Thehandle portion has an arch-like extension on a first face. The firstface is located on the domed side of the body. This face includes thedomed body portion 51 with projecting guides 52 and 53 of the cartridgeand the first face of the hand portion 61, defined along an upper edgeof improved grips 37, 38, a curved upper edge of the arch-like extension39, and the upper edge 40 which forms a border above the domed bodyportion 51 of the cartridge. A second face of the handle is opposite thedome body, and contains an inner-framed structure bounded along theradial axis of the cartridge by the two keeper elements 77, 77 and alonga longitudinal axis by the uppermost portion of the handle and theenlarged mouth 67 of the vestibule. Changes to both faces provide a moresecure lock for the stopper when engaged in position, and amuch-improved rigid support for cartridge manipulation.

The projecting part of the stopper including the probe 72 and the plug74 comprise an elastomeric material such as a thermoplastic elastomer(DYNAFLEX®), or other elastomeric material capable of forming seals withthe inlet 65 and the mouth 67, respectively. The elastomeric materialmust also be non-fluorescent and non-reactive with the sample. Thedurometer of the elastomeric material is in the range of about 60 to 100with a more preferred range of about 80 to 90 and the most preferredabout 90. The handhold 75 of the stopper and the flexible walls 76, 76are formed of the same elastomer as the probe and plug, thus making theentire stopper composed of a single elastomer. As with the parentalcartridge/stopper, there is little ability to twist the stopper handhold75 about its axis, especially after the flexible walls are securelylocked into place. To release their engagement with the keepers 77, 77,a window within the framed area of the cartridge handhold 61 allowsaccess for manual compression of the flexible walls and reversal of theengagement. Overall, this single elastomer design provides for a lowercost cartridge/stopper assembly.

As shown in FIG. 4C, the cartridge is disposed with its longitudinalaxis horizontal so that the flat land area 62 of the test chamber 63 isdisposed within the field of observation of the detection equipment.When filling the test chamber 63, the cartridge is disposed with itslongitudinal axis upright with the vestibule 66 disposed above the testchamber 63.

As shown in FIGS. 5A to 5C, a test liquid is introduced into the testchamber 63 along with any associated buffer solution. The buffersolution has a density which is less than or equal to the test liquid sothat there is a liquid interface provided between the two solutions. Thevolume of the buffer solution is sufficient to completely fill the testchamber 63. The filling operation excludes air from the test chamber 63,and any air bubbles remaining in the buffer solution will gravitateupwardly through the inlet 65 into the vestibule 66.

The construction and arrangement of the chamber insures that air bubblesare excluded from the test chamber and pressure build-up in the unit isavoided. As shown in FIGS. 5A to C, when the stopper 60 is inserted intothe vestibule, a projecting portion containing the probe 72 displacesthe buffer solution as it enters the established fill line and causesthe surface of the buffer solution to rise within the vestibule untilthe probe 72 contacts the inlet 65. As shown, the inlet 65 has a flaredmouth 88 and a cylindrical channel 89 below the flared mouth (FIG. 5B).At this point, the surface of the buffer solution is shifted upwardly toan elevated fill line. Further movement of the stopper downwardly causesthe tip of the probe 72 to enter the channel 89 of the inlet 65. Whenthe probe 72 engages the cylindrical channel 89, the probe affects afirst seal, closing communication between the test chamber 89 and thevestibule 66. Further penetration of the probe 72 into the cylindricalportion of the cylindrical channel 89 of the inlet 65 perfects the seal(FIG. 5C). Because the tip of the probe 72 closes the cylindricalchannel 89, and, in the present instance, the internal diameter of thechannel 89 of the inlet 65 is less than the internal dimensions of thechamber 63 so that the volume of buffer solution in the channel isminimal, and the engagement of the probe 72 into the channel 89 wheneffecting the first seal does not significantly increase the pressurewithin the test chamber 63. The vestibule remains open at the top untilthe plug 74 enters the mouth 67, allowing the vestibule to remain atambient pressure.

The design of the present invention may be used for any analysischamber, but it has been specifically created for analysis chambers fortesting extremely small samples having a volume of less than 1 ml. Inthe illustrated embodiment, referring to FIG. 4B, the width of thechamber 63 below the land area 62 is approximately 3 mm and the depth isapproximately 4 mm, providing a cross-sectional area in the range of 10to 14 square millimeters. The length along the longitudinal axis isapproximately 30 mm. The volume of the chamber 63 should be in the rangebetween 22 ul and 675 ul, preferably at least 315 ul. The diameter ofthe inlet 65 is in the range between 0.0381 mm and 3.18 mm, andpreferably is 2.35 mm, providing a flow area of approximately 10 squaremillimeters. Beyond the inlet 65, the vestibule flares out, in this caseto a diameter of 4.23 mm, and extends approximately 14 mm to the mouth67, which, in this case, has a width of 6.3 mm. With the plug fullyinserted, the volume of the vestibule 66 is preferably at least 95 ul.The width of the test chamber at the upper wall is slightly more thanthe diameter of the channel 89 of the inlet 656. Although not shown inthe figures, the corners around the perimeter of the upper wall arebroken or beveled, so as to avoid entrapment of any air bubblesgravitating upwardly through the chamber 63. The bevel is preferably atan angle of between 2° and 30° relative to the longitudinal axis of thechamber 63 and the inlet 65.

Further displacement of the stopper 60 allows the plug 74 at theproximal end of the stem 73 to engage in the enlarged mouth 67 of thevestibule and affect a second seal closing the upper end of thevestibule. As shown in FIGS. 6 and 7, the plug 74 is a rectangular blockto mate with the enlarged mouth 67, which is in the form of a sockethaving a complementary rectangular form. The block 74 is of the sameplastic material as the probe 72 having sufficient resiliency to effecta good seal with the enlarged mouth 67 when engaged as shown in FIG. 5C.The displacement of the plug 74 in the enlarged mouth 67 does notsubstantially increase the air pressure above the fill line 87.

The distance between the top of the cylindrical channel 89 of the inlet65 and the bottom of the socket forming the enlarged mouth 67 is lessthan the distance between the tip of the probe 72 and the bottom of theplug 74 so that there is assurance that the probe enters into thechannel 89 before the plug 74 seats against the bottom of the socket.This arrangement insures avoidance of any substantial pressure build-upin the vestibule 66. It is noted that the stem 73 has ribs 91 spacedcircumferentially there around so that the space between the ribsprovides an adequate space to accommodate the buffer solution displacedduring the penetration of the probe into the cylindrical portion 89 ofthe inlet.

With the flexible walls engaging the keeper element 77 to keep thestopper in place, air is confined within the vestibule 66 between thefirst and second seals, and the cartridge may be manipulated withoutfear of air bubbles or the like interfering with the optical analysis ofthe liquid in the test chamber 63. After being filled and capped, thecartridge may be reoriented so that its longitudinal axis is horizontalfor analysis in the detecting apparatus, as described above andillustrated in FIGS. 1 to 4, since the test chamber 63 is completelyfilled with liquid, it may be manipulated into other orientations, asmay be required by the detecting apparatus chosen by the analyst. Anybuffer solution, which is in contact with the test liquid is retainedwithin the cartridge, either in the test chamber 63 or within thevestibule 66, and there is little danger of loss of any significant partof the test liquid. The buffer solution, which overlies the test liquidin the cartridge during the filling operation, assures minimal exposureof the test liquid to air and a diminished risk of deterioration orcontamination of the test liquid.

While particular embodiments of the present invention have been hereinillustrated and described, it is not intended to limit the invention tosuch disclosure, and many changes and modifications can be envisionedtherein that may be made within the scope of the following claims.

1. An improved stopper having a probe on a projecting stem of saidstopper, said probe forming a first seal adapted to sealingly seat in aninlet and close the same, said first seal precluding air entrapmentwithin a test chamber upon being seated in said inlet, said probe havinga distal end adapted to enter said inlet of said test chamber below anestablished fill level before said first seal closes said inlet, saiddistal end having a thickness sufficient to displace liquid upwardlythrough said inlet and position the surface of said fluid sample in avestibule prior to said first seal closing said inlet and a plug on saidprojecting stem of said stopper proximal to said probe, said plug havinga position along said projecting stem such that said vestibule remainsopen to an environment until after closing of said inlet by said probethen said plug engages with an enlarged mouth on said vestibuleeffecting a second seal to close the upper end of vestibule, wherein theimprovement comprises: a. a single elastomer where the probe, the plug,and a stopper handhold are composed of said single elastomer; and b. alocking mechanism to engage with keeper elements on a cartridge, saidmechanism having flexible walls on each side of said stopper handholdsuch that when said second seal closes the vestibule said flexible wallsare compressed until they snap outwardly and allow said stopper to lockinto place, said locking mechanism resists disengagement by twisting. 2.A stopper according to claim 1 wherein said elastomer has a durometervalue in the range of approximately 60 to
 100. 3. A stopper according toclaim 1 wherein said elastomer has a durometer value in the range ofapproximately 80 to
 90. 4. A stopper according to claim 1 wherein saidelastomer has a durometer value of about 90.